Many G-protein coupled receptors involved in pain and addiction are pharmacologically and biochemically well characterized, but some orphan receptors like GPR35 and GPR55, with homology to known receptors for drugs of abuse, remain poorly characterized. GPR55 is emerging as an important target in inflammatory pain, neuropathic pain, and bone development while other studies indicate that GPR55 activation is pro-carcinogenic. GPR55 does recognize certain cannabinoid ligands, so it has been suggested to be a third cannabinoid receptor. GPR35 is an important target in pain (spinal antinociception as well as inflammatory pain), heart disease, asthma, metabolic disease, inflammatory bowel disease and cancer. Each of these areas alone is medically important and each would benefit by the use of selective agonists and antagonists to further studies in their respective animal models. However, to date, no low nanomolar potency ligands have been discovered for these receptors nor is there a radioligand developed to characterize binding. The lack of such ligands is a critical barrier to progress in this field. The goal of this proposal is to leverage our recen promising high throughput, high content screening results for GPR55 agonists and GPR35 antagonists using structure based design and cheminformatics tools to develop an SAR for selected scaffolds that leads to the identification of low nanamolar ligands that retain high receptor selectivity. We aim to discover nanomolar potency GPR55 and GPR35 ligands using a combination of structure-based design, molecular modeling, chemoinformatics, high-throughput screening and site directed mutagenesis.